The collective evidence dictates that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogenesis in conditions such as diabetic retinopathy and AMD. Investigation of VEGF action has largely focused on receptor binding events and activation of classical downstream signal transduction cascades. Work from us and others show that VEGF receptor (VEGFR) signaling is much more complex and an alternative pathway involves intracellular trafficking of VEGFRs (Fig 1). We have preliminary evidence that a) endosomal sorting, neuropilin, secretases and sumoylation appear to be key regulators of VEGFR trafficking, b) VEGFRs in the nucleus originate from an intracellular pool, c) translocation of VEGFRs and the relative levels of VEGFRs within subcellular compartments dictates angiogenic outcome and d) the VEGFR1:VEGFR2 ratio at adherens junctions (AJs) and tight junctions (TJs) changes in response to pro- and antiangiogenic factors. Based on these observations we propose the following paradigm shifting hypothesis: VEGF-driven vascular permeability and neovascularization are highly dependent on the targeted subcellular translocation of specific VEGFRs in endothelial cells and that the relative levels of VEGFRs within subcellular compartments dictates vascular permeability and angiogenic outcome. We further postulate that endothelial-specific manipulation of the VEGFR1:2 ratio through gene therapy will reduce vascular permeability and inhibit aberrant retinal and choroidal neovascularization. We will test this hypothesis through the following aims. In Aim 1, we will a) To characterizing the origin and routes of VEGFR trafficking, b) identify the nuclear targets of VEGFR1 and VEGFR2 and assess how these targets contribute to angiogenesis, c) assess how the nuclear level of VEGFRs dictates the angiogenic outcome and d) identify the mechanism by which secretases, and/or sumoylation regulate trafficking of VEGFRs. In Aim 2, we will a) determine if translocation of VEGFRs to AJs and TJs is via membrane diffusion or endosomal trafficking and b) assess how the ratio of VEGFR1 and VEGFR2 at AJs and TJs changes in response to pro- and antiangiogenic factors, the junctional binding partners involved, and how this affects permeability. In Aim 3, we will assess the outcome of endothelial-specific genetic manipulation of intracellular VEGFR levels on vascular permeability and retinal and choroidal angiogenesis in mice. The proposed studies will significantly advance our mechanistic understanding of aberrant ocular angiogenesis and will identify new potential therapeutic targets for the inhibition of pathological ocular angiogenesis.